This competing renewal Program Project Grant application requests funds to continue studies on the molecular mechanisms of HAD, with a strong new focus on the role of astrocytes in HAD pathogenesis. Significant progress has been made during the last funding cycle due to synergistic collaborations among PPG investigators and shared expertise, and resulting in more than 50 publications. Among major advances, we demonstrated that primary astrocytes are capable of efficient HIV replication in culture, identified a reservoir of HIV-1 DNA in brain astrocytes by LCM, determined that HIV-1 and TNF-cc inhibit astrocyte glutamate transport through down modulation of transporters EAAT1 and EAAT2; cloned and characterized EAAT1 and EAAT2 promoters and the EAAT2 3'-UTR, and identified some of the cis-response elements and cellular factors involved. A set of new transcripts in astrocytes also modulated by HIV-1 was revealed by large scale and fine scale analysis conducted in parallel. The xenogeneic model of HIV-1 dementia in SCID mice was further developed to elucidate both innate and adaptive immune responses in the brain. New methods in neural imaging have permitted the assessment of monocyte-macrophage migration through the brain and contribution of HIV infected microglial cells to HAD pathogenesis. Based on these findings we hypothesize that HIV infected astrocytes and monocyte-derived cells act in tandem to facilitate HAD pathogenesis through a combination of diminution of neuro-protective and neuro-regulatory functions of astrocytes, neurotoxic insults by macrophage products, and increase in deleterious immune responses in the brain mediated by both cell types. This hypothesis will be tested in the present program by a broad, coordinated, and thorough evaluation of the effects of HIV and infected macrophages on glial cell biology, including glutamate transport and brain-homeostatic functions, and impact of functionally-impaired astrocytes on the metabolic and functional integrity of neurons in culture and in vivo. The proposed program will be conducted by an established team of long-term collaborators with complementary expertise in neurovirology, molecular biology, immunology, neuropathology, and animal models of HAD (who directed Projects 1-3 in the current PPG) joined by a neurobiologist specializing in in vitro models of neuronal function (new Project 4). Project 1 (Volsky), will examine HIV-1 induced changes in cellular interactions by defining the metabolic pathways and gene products affected in primary astrocytes by HIV-I and their impact upon neuronal function. Project 2 (Fisher) will study specific regions and mediators of alteration in glutamate transporter EAAT2 transcription in the context of neurodegeneration emphasizing the role of the novel astrocyte gene AEG-I. Project 3 (Gendelman) will investigate the interactions among HIV-infected macrophages and infected astrocytes leading to neuropathogenesis in the SCID HAD model; and Project 4 (Gelbard) will determine the link between astrocyte gene products and impairment of specific, essential neuronal functions including neurite extension and synapse formation using a compartmentalized astrocyte-neuron co-culture system. The projects will be supported by a Virology/Cell Genetics Core 1 (Potash), Neuropathology Core 2 (Sharer), and Administrative Core 3 (Volsky). These studies should lead to better understanding of HIV-mediated neuropathogenesis and to development of novel treatments based on countering astrocyte dysfunction in HIV disease.